AGUA

In this study, we exposed human A549 lung epithelial cells to formaldehyde using an in vitro exposure system that physically replicates in vivo human lung gas exposures (Bakand et al. 2005). It is important to note that A549 cells are carcinoma cells that exhibit differences in certain signaling compared to non-cancerous cells. For instance, A549 cells are enriched for Nrf2

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detoxifying pathways and are more resistant to apoptosis in comparison to normal cells (Kweon et al. 2006). While we recognize that A549 cells may not completely mimic normal lung cell response, there are several advantages to using these cells for air toxicant studies. For example, when exposed to gases at an air-liquid interface, A549 cells secrete enough surfactant to mimic airway surface tension (Blank et al. 2006). As a result, A549 cells are routinely used to study the effects of environmental air exposures (Doyle et al. 2004; Doyle et al. 2007; Jaspers et al. 1997; Sexton et al. 2004), including formaldehyde (Quievryn et al. 2000; Speit et al. 2008; Speit et al. 2010). A549 cells have also shown the same sensitivity and removal efficiency towards formaldehyde-induced DNA protein crosslinks as primary human nasal epithelial cells (Speit et al. 2008).

Our microarray analysis revealed that formaldehyde exposure resulted in the down- regulation of 89 miRNAs. It was interesting that all of the modulated miRNAs were down- regulated by formaldehyde exposure. This general trend of miRNA down-regulation has been observed in rat lung cells exposed to cigarette smoke (Izzotti et al. 2009), as well as in multiple tumor cell types, including lung cancer, breast cancer, and leukemia (Lu et al. 2005).

We focused a detailed analysis on the four most significantly down-regulated miRNAs, as determined through microarray analysis and RT-PCR: miR-33, miR-330, miR-181a, and miR- 10b. These miRNAs have been studied, to some extent, and knowledge about their regulation and association to disease is growing. For example, miR-33 shows decreased expression levels in tissues from patients with lung carcinomas (Yanaihara et al. 2006). Also, miR-330 expression has been measured at significantly lower levels in human prostate cancer cells when compared against nontumorigenic prostate cells (Lee et al. 2009). Furthermore, miR-330 has been suggested to act as a tumor suppressor by regulating apoptosis of cancer cells (Lee et al. 2009). In addition, miR-10b shows altered expression levels within breast cancer tissue, and is one of the most consistently dysregulated miRNAs able to predict tumor classification (Iorio et al. 2005; Ma et al. 2007). These findings suggest that miR-33, miR-330, and miR-10b may influence cellular disease state, specifically related to cancer.

Formaldehyde exposure also altered the expression level of miR-181a, which has known associations with leukemogenesis (Marcucci et al. 2009). The specific link between formaldehyde exposure and leukemia is currently debated, as numerous epidemiological studies show evidence for possible association to this disease (Hauptmann et al. 2009; Pinkerton et al.

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2004; L Zhang et al. 2010), as well as against it (Bachand et al. 2010; Marsh et al. 2004). However, it is important to note that our study evaluates miRNA expression in lung cells, which likely differ from leukemia target cells’ responses to formaldehyde exposure, or exposure to formaldehyde’s metabolic products. Nevertheless, it is worth highlighting the observation of the dysregulation of miR-181a upon exposure to formaldehyde.

To expand our analysis, we used a systems biology approach to understand the potential biological implications of the miRNA expression changes induced by acute formaldehyde exposure. For this analysis, we used a stringent computational matching approach to identify predicted mRNA targets for miR-33, miR-330, miR-181a, and miR-10b. The identified mRNA targets were used to construct associated molecular networks and were analyzed for their known involvement in signaling pathways and biological functions. The identified networks showed enrichment for various canonical pathways including nuclear factor kappa-B (NFκB) and interleukin-8 (IL-8) signaling. Although very few predicted targets overlapped between the four miRNAs, proteins involved with cancer mechanisms including that of the NFκB pathway were found within the miRNA target networks. Importantly, NFκB has clear links to inflammation and cancer development (Karin et al. 2005). Also related to inflammation, IL-8-related signaling molecules were present in the miRNA target networks. Previous studies have shown IL-8 release in lungs cells representing inflammatory response after exposure to other air pollutants (Jaspers et al. 1997; Sexton et al. 2004). In addition, investigations have shown increased IL-8 levels in lungs of patients with diseases such as acute lung injury (McClintock et al. 2008), adult respiratory distress syndrome (Jorens et al. 1992), and asthma (Bloemen et al. 2007). Inflammation is a recognized formaldehyde-induced response, as formaldehyde is known to irritate the respiratory system (Tuthill 1984) and increase asthmatic response (Rumchev et al. 2002; Wieslander et al. 1997). Our findings suggest that the canonical pathways associated with formaldehyde-induced miRNA alterations may affect the regulation of biological pathways associated with various disease states, including cancer and inflammation.

As a method to further verify our results, we compared the protein levels of cytokine interkeukin-8 (IL-8) in formaldehyde-exposed cells versus mock-treated controls. We found that, indeed, IL-8 showed significantly increased protein expression levels in the formaldehyde- exposed cells. These results support our findings that IL-8 signaling is altered in lung cells exposed to formaldehyde. Interestingly, IL-8 levels are also increased in formaldehyde-exposed

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lung cells after pre-sensitization to tumor necrosis factor alpha (TNFα) (Persoz et al. 2010). TNFα is a proinflammatory mediator shown to have increased levels upon exposure to formaldehyde (Bianchi et al. 2004). Our network analyses suggest that cytokine signaling may be altered through changes in miRNA expression levels. Supporting this is a recent study that shows modifications to miRNAs may influence the expression of cytokines, including IL-6 and IL-8 (Jones et al. 2009). Future research will test whether the observed miRNA expression changes are directly associated with IL-8 signaling.

In an effort to gain further understanding of formaldehyde’s effects on gene expression, we compared our results with those of an existing genomics database (e.g. mRNA) from a study that evaluated human lung cells exposed to formaldehyde (Li et al. 2007). Using the predicted targets in our most significant miRNA networks, we found the following genes overlap with the existing database: BDNF, BMPR2, CACNA1C, CSNK1D, HMGA2, HSF2, HSPH1, and PIM1. These genes have been shown to play a role in various diseases. For example, BDNF, or brain- derived neurotrophic factor, modulates neurogenesis after injury to the central nervous system (Ming et al. 2005). CSNK1D, or casein kinase 1 delta, has been identified as up-regulated in breast cancer tissue (Abba et al. 2007). HMGA2, or high mobility group AT-hook 2, is oncogenic in many cells, including lung carcinoma cells, and is regulated by the tumor-suppressive miRNA let-7 (Lee et al. 2007). Lastly, PIM1, or Pim-1 oncogene, is found at increased levels within prostate cancer tissue (Dhanasekaran et al. 2001). Network analysis of all formaldehyde- responsive genes identified through the Li et. al. (2007) study revealed significant associations with cancer, inflammation, and endocrine system regulation, which also overlap with our findings. These genes are therefore linked with formaldehyde-induced changes in miRNA abundance as well as mRNA alterations, and they are related to a diverse range of cellular responses including tumorigenesis.

In conclusion, our study provides evidence of a potential mechanism that may underlie the cellular effects induced by formaldehyde, namely the modification of miRNA expression. We identify a set of 89 miRNAs that are dysregulated in human lung cells exposed to formaldehyde. Mapping the most significantly changed miRNAs to their predicted transcriptional targets and their network interactomes within the cell reveals the association of formaldehyde exposure to inflammatory response pathways. We also validate our findings by: (1) performing RT-PCR; (2) integrating our predicted networks with known formaldehyde-

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induced mRNA expression changes; and (3) examining protein expression changes of a key inflammatory response mediator, IL-8. Future research will investigate whether the expression levels of these miRNAs may serve as potential biomarkers of formaldehyde exposure in humans. Such biomarkers can be utilized to better monitor human exposure to environmental toxicants and relate them to health effects. Based on our findings, we believe that miRNAs likely play an important role in regulating formaldehyde-induced gene expression and may represent a possible link between exposure and disease.

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